AMPK is well established as a sensor and regulator of cellular energy homeostasis. Allosteric activation of this kinase due to rising AMP levels occurs in states of cellular energy depletion. The resulting serine/threonine phosphorylation of target enzymes leads to an adaptation of cellular metabolism to low energy state. The net effect of AMPK activation induced changes is inhibition of ATP consuming processes and activation of ATP generating pathways, and therefore regeneration of ATP stores. Examples of AMPK substrates include acetyl-CoA carboxylase (ACC) and HMG-CoA reductase. Phosphorylation and therefore inhibition of ACC leads to simultaneous decrease in fatty acid synthesis (ATP-consuming) and increase in fatty acid oxidation (ATP-generating). Phosphorylation and resulting inhibition of HMG-CoA reductase leads to a decrease in cholesterol synthesis. Other substrates of AMPK include hormone sensitive lipase, glycerol-3-phosphate acyltransferase, malonyl-CoA decarboxylase.
AMPK is also involved in the regulation of liver metabolism. Elevated glucose production by the liver is a major cause of fasting hyperglycemia in type 2 diabetes (T2D). Gluconeogenesis in the liver is regulated by multiple enzymes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase—G6Pase. Activation of AMPK suppresses the transcription of theses genes in hepatoma cells.
AMPK activation also down-regulates gluconeogenesis acting on some other genes expression. These effects may be due to its ability to down-regulate key transcription factors such as SREBP-1c, ChREBP, or HNF-4alpha or to direct phosphorylate transcriptional coactivators such as p300 or TORC2.
AMPK is considered as an attractive candidate for contraction-induced skeletal muscle glucose uptake because it is activated in parallel with elevation in AMP and a reduction in creatine phosphate energy stores. Furthermore, AICAR-induced activation of AMPK increases glucose uptake concomitantly with glucose transporter 4 (GLUT4) fusion with plasma membrane. Over-expression of an alpha2 kinase dead subunit in skeletal muscle abolishes AICAR, but partially impairs contraction-stimulated glucose uptake. These findings suggest that additional pathways mediate contraction induced glucose uptake, whereas it is clear that AMPK mediates the effects of AICAR on glucose uptake.
Despite extensive studies on upstream stimuli that activate AMPK, investigation on the downstream substrate(s) of AMPK-mediated glucose uptake is lacking. More recent reports revealed that Akt substrate of 160 kDa (AS160) is an important substrate downstream of Akt that is involved in insulin-stimulated glucose uptake. In addition to insulin, contraction and activation of AMPK by AICAR is associated with increased phosphorylation of AS160 in rodent skeletal muscle. Phosphorylation of AS160 is impaired or abolished in skeletal muscle from AMPK a2 knockout, g3 knockout, and a2-kinase dead mice in response to AICAR treatment. This corroborates findings of impaired AICAR-stimulated glucose uptake in skeletal muscle of such mice. Therefore, AS160 appears to be a downstream target of AMPK in mediating glucose uptake in skeletal muscle.
Taken together, all these metabolic effects evidence that AMPK suppresses liver gluconeogenesis and lipid production, while decreasing hepatic lipid deposition via increased lipid oxidation, thus improving the glucose and lipid profiles in T2D.
More recently, involvement of AMPK in the regulation of not only cellular but also whole body energy metabolism has become apparent. It was shown that the adipocyte-derived hormone leptin leads to a stimulation of AMPK and therefore to an increase in fatty acid oxidation in skeletal muscle. Adiponectin, another adipocyte derived hormone leading to improved carbohydrate and lipid metabolism, has been shown to stimulate AMPK liver and skeletal muscles. The activation of AMPK in these circumstances seems independent of increasing cellular AMP levels but rather due to phosphorylation by one or more upstream kinases yet to be identified.
Based on the knowledge of the above-mentioned consequences of AMPK activation, deep beneficial effects would be expected from in vivo activation of AMPK. In liver, decreased expression of gluconeogenic enzymes would be expected to reduce hepatic glucose output and improve overall glucose homeostasis; both direct inhibition and/or reduced expression of key enzymes in lipid metabolism would be expected to increase glucose uptake and fatty acid oxidation with resulting improvement of glucose homeostasis and, due to a reduction in intra-myocyte triglyceride accumulation, to improved insulin action. Finally, the increase in energy expenditure should lead to a decrease in body weight. The combination of these effects in the metabolic syndrome would be expected to significantly reduce the risk of developing cardiovascular diseases. Several studies in rodents support this hypothesis. Until recently, most in vivo studies relied on AICAR AMPK activator, a cell permeable precursor of ZMP. ZMP, a structural analogue of AMP, acts as an intracellular AMP mimic and, when accumulated to high enough levels, is able to stimulate AMPK activity. However, ZMP also acts as an AMP mimic in the regulation of other enzymes, and is therefore not a specific AMPK activator. Several in vivo studies have demonstrated beneficial effects of both acute and chronic AICAR administrations in rodent models of obesity and type 2 diabetes. For example, 7 week AICAR administration in the obese Zucker (fa/fa) rat leads to a reduction in plasma triglycerides and free fatty acids, an increase in HDL cholesterol, and a normalisation of glucose metabolism as assessed by an oral glucose tolerance test (Minokoshi Y. et al. “Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase”, Nature, 415, 339, 2002)). In both ob/ob and db/db mice, 8 day AICAR administration reduces blood glucose by 35% (Halseth A. E. et al. “Acute and chronic treatment of ob/ob and db/db mice with AICAR decreases blood glucose concentrations”, Biochem. Biophys. Res. Comm., 294, 798 (2002)). In addition to AICAR, it was found that the diabetes drug metformin can activate AMPK in vivo at high concentrations, although it has to be determined to what extent its antidiabetic action relies on this activation. As with leptin and adiponectin, the stimulatory effect of metformin is indirect via activation of an upstream kinase. More recently, a small molecule AMPK activator has been described. This direct AMPK activator, named A-769662, is a thienopyridone and induces in vivo a decrease in plasma levels of glucose and triglycerides.
In addition to pharmacological intervention, several transgenic mice models have been developed in the last years, and initial results are currently becoming available. Expression of dominant negative AMPK in skeletal muscle of transgenic mice demonstrated the effect of AICAR on stimulation of glucose transport is dependent on AMPK activation, and therefore likely not caused by non-specific ZMP effects. Similar studies in other tissues will help to further define the consequences of AMPK activation. It is expected that pharmacological activation of AMPK will have benefits in the metabolic syndrome with improved glucose and lipid metabolisms and reduction in body weight. In order to qualify a patient as having metabolic syndrome, three out of the five following criteria must be met:                1) elevated blood pressure (above 130/85 mmHg),        2) fasting blood glucose above 110 mg/dl,        3) abdominal obesity above 40″ (men) or 35″ (women) waist circumference, and blood lipid changes as defined by        4) increase in triglycerides above 150 mg/dl or        5) decrease in HDL cholesterol below 40 mg/dl (men) or 50 mg/dl (women).        
Therefore, the combined effects that may be achieved through activation of AMPK in a patient who is qualified as having metabolic syndrome would raise the interest of this target.
Stimulation of AMPK has been shown to stimulate expression of uncoupling protein 3 (UCP3) skeletal muscle and might therefore be a way to prevent from damage from reactive oxygen species. Endothelial NO synthase (eNOS) has been shown to be activated through AMPK mediated phosphorylation, therefore AMPK activation can be used to improve local circulatory systems.
AMPK has a role in regulating the mTOR pathway. mTOR is a serine/threonine kinase and is a key regulator of protein synthesis. To inhibit cell growth and protect cells from apoptosis induced by glucose starvation, AMPK phosphorylates TSC2 at Thr-1227 and Ser-1345, increasing the activity of the TSC1 and TSC-2 complexes to inhibit m-TOR. In addition, AMPK inhibits mTOR action by phosphorylation on Thr-2446. Thus, AMPK indirectly and directly inhibits the activity of mTOR to limit protein synthesis. AMPK may also be a therapeutic target for many cancers that have constitutive activation of the PI3K-Akt signaling pathway. Treatment of various cancer cell lines by AICAR attenuated the cell proliferation both in in vitro and in vivo studies. Two reports link the treatment with metformin with a lower risk of cancer in diabetic patients.
Activation of AMPK by AICAR has been shown to reduce expression of the lipogenic enzymes FAS and ACC, resulting in suppression of proliferation in prostate cancer cells. Many cancer cells display a markedly increased rate of de novo fatty acid synthesis correlated with high levels of FAS. Inhibition of FAS suppresses cancer cell proliferation and induces cell death. Thus, AMPK activation and inhibition of FAS activity is a clear target for pharmacological therapy of cancers.
In some publications it has been described that AICAR as an AMPK activator exerts anti-inflammatory effects. It has been observed that AICAR attenuates the production of proinflammatory cytokines and mediators, AICAR in rat model and in vitro attenuates EAE progression by limiting infiltration of leucocytes across blood brain barrier (BBB) and it has been suggested recently that AMPK activating agents act as anti-inflammatory agents and can hold a therapeutic potential in Krabbe disease/twitcher disease (an inherited neurological disorder).